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Cite this: Dalton Trans., 2012, 41, 6726
www.rsc.org/dalton PAPER
Synthesis, spectroscopic characterization, photochemical and photophysical
properties and biological activities of ruthenium complexes with mono-
and bi-dentate histamine ligand†
Carolina R. Cardoso,a Inara de Aguiar,a Mariana R. Camilo,a Márcia V. S. Lima,a Amando S. Ito,b
Maurício S. Baptista,c Christiane Pavani,c Tiago Venâncioa and Rose M. Carlos*a
Received 8th November 2011, Accepted 19th March 2012
DOI: 10.1039/c2dt12136k
The monodentate cis-[Ru(phen)2(hist)2]
2+ 1R and the bidentate cis-[Ru(phen)2(hist)]
2+ 2A complexes
were prepared and characterized using spectroscopic (1H, (1H–1H)COSYand (1H–13C)HSQC NMR,
UV-vis, luminescence) techniques. The complexes presented absorption and emission in the visible
region, as well as a tri-exponential emission decay. The complexes are soluble in aqueous and
non-aqueous solution with solubility in a buffer solution of pH 7.4 of 1.14 × 10−3 mol L−1 for (1R + 2A)
and 6.43 × 10−4 mol L−1 for 2A and lipophilicity measured in an aqueous–octanol solution of −1.14 and
−0.96, respectively. Photolysis in the visible region in CH3CN converted the starting complexes into
cis-[Ru(phen)2(CH3CN)2]
2+. Histamine photorelease was also observed in pure water and in the presence
of BSA (1.0 × 10−6 mol L−1). The bidentate coordination of the histamine to the ruthenium center in
relation to the monodentate coordination increased the photosubstitution quantum yield by a factor of
3. Pharmacological studies showed that the complexes present a moderate inhibition of AChE with an
IC50 of 21 μmol L
−1 (referred to risvagtini, IC50 181 μmol L
−1 and galantamine IC50 0.006 μmol L
−1)
with no appreciable cytotoxicity toward to the HeLa cells (50% cell viability at 925 μmol L−1).
Cell uptake of the complexes into HeLa cells was detected by fluorescence confocal microscopy. Overall,
the observation of a luminescent complex that penetrates the cell wall and has low cytotoxicity, but is
reactive photochemically, releasing histamine when irradiated with visible light, are interesting features for
application of these complexes as phototherapeutic agents.
Introduction
Histamine (hist) has been broadly established as having impor-
tant roles in mammalian physiology, for example, as a bioregula-
tory agent in vasodilation and neuronal signaling.1,2 Moreover,
histamine has shown encouraging anti-tumor and anti-metastatic
properties3,4 and implications for Alzheimer’s disease therapy.5,6
Unfortunately, the limiting factor is the histamine intolerance.7
In this context, ruthenium–polypyridine complexes can be seen
as useful delivery agents of histamine8 and, in particular, are
promising for the photochemical delivery of hist to desired
physiological targets.
These complexes are also interesting for research tools
because the coordination to the Ru center can occur through
either N1 or N3 of the imidazole ring giving rise to the adjacent
(1A) and remote (1R) isomers. Furthermore, due to the rotation
of the CH2CH2NH2 side chain of the imidazole ring of the hista-
mine, two possible conformations of the 1A and 1R isomers,
the trans and gauche conformers, may coexist in the solution.9
The gauche conformer is expected to be more stable because
of the proximity of the NH2 group to the acid proton of the NH
imidazole and for this reason it is expected to be the major
species. For the 1A isomer, the combination of lower stability of
the trans conformer associated to higher lability of the Ru–N3
bond facilitates the formation of the bidentate Ru–hist complex.
Scheme 1 shows the possible modes of coordination of hista-
mine to the metal center Ru(II).
Having this background, herein we describe the synthesis of a
ruthenium–polypyridine complex containing histamine in an
aqueous solution, and evidence is presented for a monodentate
and bidentate coordination mode to the ruthenium center. In
addition, we also report the synthesis, in an aqueous basic sol-
ution, of the analogous complex having only the bidentate co-
ordinated histamine, as well as a comparison of the structures
obtained using NMR (1H, (1H–1H)COSY and (1H–13C)HSQC)
spectroscopy. The photophysical and photochemical properties
of the complexes are also reported.
†Electronic supplementary information (ESI) available: NMR spectro-
scopic data. See DOI: 10.1039/c2dt12136k
aDepartamento de Química, Universidade Federal de São Carlos,
CP 676, CEP 13565-905 São Carlos-SP, Brazil
bFaculdade de Filosofia Ciências e Letras de Ribeirão Preto-,
Universidade de São Paulo, Brazil
cDepartamento de Bioquímica, Instituto de Química, Universidade de
São Paulo, São Paulo, Brazil
6726 | Dalton Trans., 2012, 41, 6726–6734 This journal is © The Royal Society of Chemistry 2012
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To address the biological activity of these complexes at the
physiological medium, we investigated their aqueous solubility,
hydrophobicity as well as in vitro inhibitory effect on cell pro-
liferation and cell uptake into the HeLa cells line. The kinetics of
the interaction of the complexes with acetylcholinesterase was
also evaluated.
Results and discussion
1. Preparation, structure and electronic characteristics
The reaction of the complex cis-RuCl2(phen)2 with 2 equiv. of
histamine at a H2O : EtOH (1 : 1) ratio led to the formation of a
monodentate and bidentate complex and a small amount of the
conformers of these complexes. All attempts to separate the
complexes and synthesize only the monodentate complex have
failed so far, even when an excess of histamine was used.
However, the major product of the synthesis is the 2A complex,
possibly because a 6 membered ring is formed when the terminal
NH2 coordinates with a metallic center. The pure bidentate
complex, 2A, was obtained by the reaction of cis-RuCl2(phen)2
with the histamine ligand in a 2 : 1 ratio in an aqueous solution,
pH 10. The coordination modes of the histamine to the ruthe-
nium center were confirmed by NMR spectroscopic measure-
ments. The monodentate complex was found to have two
histamine ligands coordinated cis- to one another by the N1
nitrogen atom of the imidazole ring giving rise to the remote
isomer (1R), whereas in the bidentate complex the chelation
occurred through the N3 nitrogen atom of the imidazole ring and
the N of the amine terminal NH2 of the CH2CH2NH2 side chain
producing the adjacent isomer, 2A. The assignments were made
on the basis of 1H, (1H–1H) COSY and (1H–13C)HSQC experi-
ments, Fig. 1, Scheme 1, Table 1 and Fig. S1, S2, S3, S4.†
(a) 1H NMR. The analysis of the 1H-NMR spectra of both
mono and bidentate complexes revealed the differences between
the signals from the imidazole ring and those of the
CH2CH2NH2 side chain of the imidazole ring of both com-
pounds. The H1a, H1a′ and H2a, H2a′ (CH) ring protons of imi-
dazole of the monodentate complex, 1R, showed signals at 7.40
and 6.57 ppm while in the bidentate complex, 2A these signals
showed a slight upfield effect to 7.01 and 6.10 ppm as is shown
Scheme 1 Possible structures resulting from the coordination of histamine on the {(phen)2Ru}
2+ fragment.
This journal is © The Royal Society of Chemistry 2012 Dalton Trans., 2012, 41, 6726–6734 | 6727
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in Table 1 and Fig. 1. Most significant is the fact that in the
monodentate complex, the amine terminal NH2 of the
CH2CH2NH2 side chain of the imidazole ring is probably
involved in intra-molecular interaction with the NH(imidazole
ring), as observed for free histamine.10 This interaction could be
affecting the signal of the proton from the imidazole NH group
(10.35 ppm), which is not present in the 1H NMR spectrum of
the monodentate complex.
The corresponding shifts in the H3a and H4a (CH2) protons
of the CH2CH2NH2 side chain of the imidazole ring from 2.48
and 2.70 ppm in the monodentate to 2.74 and 3.00 ppm in the
bidentate coordination are indicative of the decrease in electron
density at these sites due to delocalization of the negative charge
to the Ru center.
For both complexes, the phenanthroline aromatic ring protons
show signals between 9.49 and 7.39 ppm. A characteristic of the
monodentate complex is that the two phen ligands lie in magne-
tically equivalent positions. Thus, due to the same chemical
environment, the proton signals H3, H3′ and H4, H4′ are ident-
ified at the same position. In contrast, for the bidentate complex,
the H3 and H4 protons near to the NH2 group appear at
9.49 ppm and 8.08 ppm, whereas the H3′ and H4′ protons near
to the imidazole ring are identified at 8.71 ppm and 7.79 ppm,
respectively. Owing to the combination of inductive and steric
effects, the protons H3 and H4 experience an upfield effect with
respect to those in the monodentate complex.
The spectroscopic techniques used to characterize the mixture
(1R + 2A) synthesized in this work showed the presence of five
species in the solution. We were not able to separate and isolate
these species, however on the basis of integrating the 1H NMR
signals we identified these species as 2A (bidentate, 61%), 1R
(monodentate, 30%) and three conformers, which originated
from monodentate histamine coordination (9%). The presence of
the complexes 1R and 2A is clearly observed by the NOE-NMR
experiments, as shown in and discussed in the next section. The
conformers were observed only in the 1H NMR spectrum of
the mixture (1R + 2A) as less intense signals in the region of the
imidazole ring and in the phenanthroline ligand.
We were able to prepare and isolate the 2A complex using a
basic medium (pH = 10), because in this condition the NH2 is
found in a deprotonated form and, consequently, the bidentate
coordination was the preferred form. It can be observed that the
bidentate coordination of the histamine ligand does not enable
the CH2CH2NH2 group to rotate and as a consequence, the
minority conformers obtained in the mixture synthesis are not
observed by 1H NMR.
(b) 1D-nOesy experiments. Further structural insight into the
bidentate complex formation came from the 1D-Noesy experi-
ment, Fig. 2 and 3. Irradiation at 7.00 ppm resonance in the
bidentate complex leads to an increase in the intensity of the
signals at 2.74 and 3.00 ppm, related to the CH2CH2NH2 side
chain of the imidazole ring of the histamine ligand, indicating
that these protons are spatially close to each other, but very
distant from the phenanthroline ligand because no effect is
observed in the phenanthroline signals, Fig. 2a. In contrast, the
irradiation of 6.10 ppm proton signals caused nOe in the phenan-
throline signals at 7.79 and 8.71 ppm, which are related to H4′
and H3′ protons. nOe for the terminal NH2, in 2.26 ppm, from
the CH2CH2–NH2 group can also observed, which is spatially
closed to the irradiated signals, Fig. 2b.
The 1D-nOesy experiments were also performed for the
monodentate complex by irradiating at 7.40 ppm and 6.57 ppm
resonances, Fig. 3. The irradiation of 7.40 ppm only shows nOe
in the phenanthroline signals (9.39 and 8.04 ppm), related to H3
and H4 protons, Fig. 3a. To note, the irradiation of 7.4 ppm
signal is not very selective, because the H1a from the 1R
complex and also the H9 proton from the 2A complex are
affected. The result of this non-selective irradiation leads to the
observation of signals in 7.83 and 8.38 ppm, related to protons
H10 and H8 from the 2A complex and a nOe at 2.70 ppm
related to protons H4a. By irradiating the 6.57 ppm signal, a nOe
can be detected at 2.48 ppm, which is related to H3a protons
from a CH2CH2NH2 side chain of the imidazole ring of the his-
tamine, a nOe for H3 and H4 protons from phenanthroline (9.39
and 8.04 ppm, respectively) and a nOe for the H1a proton
(7.41 ppm) from the imidazole ring of the other ligand, Fig. 3b.
The NMR experiments show that monodentate coordination
of the histamine ligand occurs via Ru–N1(imidazole ring), i.e., it
forms a stable complex with a remote isomer, 1R, whereas the
Table 1 1H NMR (1D and 2D-COSY) chemical shift of 2A and 1R in
CD3CN
(1R) (2A)
H δH (mult, Hz) H δH (mult, Hz)
3,3′ 9.39 (dd; 1.0/5.2)(2H) 3 9.49 (dd; 1.1/5.1)(1H)
5,5′ 8.65 (dd; 1.2/8.2)(4H) 3′, 5, 5′ 8.71 (m) (3H)
4,4′ 8.04 (dd; 5.2/8.1) 4 8.08 (dd; 5.2/8.1) (1H)
4′ 7.79 (dd; 1.2/5.2) (1H)
6,6′ 8.09 (d; 8.8) (2H) 6,6′ 8.00 (dd; 8.8) (1H)
7,7′ 8.18 (d; 8.8) (2H) 7,7′ 8.16 (d; 8.8) (1H)
8,8′ 8.37 (dd; 0.8/7.6)(3H) 8,8′ 8.38 (dd; 0.8/8.2)(2H)
9,9′ 7.39 (dd; 5.2/8.1)(4H) 9,9′ 7.41 (m; 5.3/8.2)(2H)
10,10′ 7.79 (dd; 1.2/5.2)(1H) 10,10′ 7.83 (dd; 1.2/5.2)(1H)
1a 7.40 (s) (2H) 1a 7.00 (s; 1.2)(1H)
2a 6.57 (s) (2H) 2a 6.10 (d; 1.2) (1H)
3a 2.48 (t; 6.6) 3a 3.00 (q; 4.4)
4a 2.70 (t; 6.6) 4a 2.74 (m)
NH 10.35
Fig. 1 1H NMR spectrum of (1R + 2A) and 2A in CD3CN.
6728 | Dalton Trans., 2012, 41, 6726–6734 This journal is © The Royal Society of Chemistry 2012
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bidentate histamine form leads to the adjacent isomer, 2A,
Ru–N3(imidazole ring).
The structures and isomeric forms of the two complexes were
further supported by DFT optimized geometries of 1R, 1A and
2A (see below).
Complex 1R was identified but could not be separated from
2A. For this reason its spectroscopic, photochemical and photo-
physical properties were evaluated by comparing the results
obtained in the mixture (1R + 2A) with those of 2A obtained
separately.
(c) Absorption and emission properties. The thermal stability
of these complexes in the dark was proved by the absence of
changes in the electronic absorption spectra in all solvents tested
over a period of 24 hours. The UV-vis spectra of the complexes,
obtained in acetonitrile, show intense bands at higher energy
associated mainly with π–π* electronic transitions (<350 nm)
and a broad absorbance at 400–600 nm, characteristic of MLCT
processes. Excitation in the visible region band, in acetonitrile
and at room temperature, produces a relatively intense emission
at 640 nm. The excitation spectrum (λem = 640 nm) resembles
the absorption spectrum and the intensity of emission is solvent
dependent: it is higher in CH2Cl2 and lower in CH3CN.
The emission quantum yields in CH3CN for (1R + 2A) and 2A
complexes are similar but lower11 than [Ru(phen)3]
2+ having
0.010 values. The fit of the emission decay profiles measured in
the CH3CN solution to a monoexponential curve resulted in high
values of reduced-χ2 values (1.864 for (1R + 2A) and 1.670 for
2A). Furthermore, the plot of residuals showed a non-random
distribution of the differences between the fitted curve and
the experimental data (Fig. 4A). A best fit was obtained with a
bi-exponential curve, with reduced-χ2 values close to 1.0 and
a random distribution of residuals (Table 2 and Fig. 4B).
The decays were described by a long lifetime, 248.5 ns for
(1R + 2A) and 137.3 ns for 2A, and a short lifetime component,
35.5 ns for (1R + 2A) and 20.5 ns for 2A and thecontribution
from the long lifetime accounts for 98.5% of the total emission.
The normalized pre-exponential factor of the long lifetime was
equal to 0.90 in both samples, meaning that there is a relative
ratio of 0.9 : 0.1 between the initial concentrations of the long
lifetime and the short lifetime emitting species. The emission
lifetimes of both complexes are shorter than that for
[Ru(phen)3]
2+ (500 ns),11 and we observe that while the relative
populations of the long and the short lifetime species are the
same in both complexes, the lifetime values increase by a factor
of 1.9 in (1R + 2A) compared to 2A.
Fig. 2 1D-nOesy spectrum of 2A in CD3CN with irradtion at: (A) 7.00 ppm and (B) 6.10 ppm.
Fig. 3 1D-nOesy spectrum of 1R in CD3CN with irradtion at: (A) 7.40 ppm and (B) 6.57 ppm.
This journal is © The Royal Society of Chemistry 2012 Dalton Trans., 2012, 41, 6726–6734 | 6729
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The UV-vis and luminescent characteristics of the complexes
are maintained in pure water and in an aqueous buffer solution
(Tris/HCl, pH 7.4) in the absence and presence of bovine serum
albumin (see below). This is relevant in terms of developing
drug delivery systems considering that serum albumin has been
investigated as the binding and transport protein of metallodrugs
for their targets in physiologic medium.12–15
2. Continuous photolysis
The complexes are photoreactive and the changes in their elec-
tronic absorption and emission spectra in 420 nm light
irradiation are consistent with histamine release and the for-
mation of a solvated complex, Fig. 5 and 6. The maximum
wavelength of the photoproduct reproduces the maxima absorp-
tion of the cis-[Ru(phen)2(CH3CN)2]
2+ complex, synthesized
thermally (λmax = 420 nm; εmax = 8700 mol
−1 L cm−1) and for
which the maximum absorption matches the values reported in
the literature.16 Furthermore, the signals at 9.77 ppm in the 1H
NMR obtained after 40 min irradiation of the complexes corre-
spond to the cis-[Ru(phen)2(CD3CN)2]
2+ ion complex17
(Fig. 5C and 6C).
The quantum yields, at 420 nm irradiation, of the histamine
photosubstitution (Φsubs) were calculated on the basis of
depletion in intensity of the signals at 6.56 ppm (1R) and
6.1 ppm (2A) of the imidazole ring proton, using the method
proposed in the literature18 (Fig. 4C and 5C). The Φsubs for
complex 2A (0.092) was higher than that for complex 1R,
0.0298. This is consistent with the weaker Ru–NH2-
(CH2CH2NH2) bond in complex 2A compared to the Ru–N-
(imidazole ring) bond present in 1R.
When the photolysis was carried out in an aqueous solution,
no drastic changes in the absorption spectra were observed
because the photoproduct cis-[Ru(phen)2(H2O)2]
2+ (λmax =
469 nm; εmax = 10 700 mol
−1 L cm−1)19 presents an intense
absorption band at the same region of the starting complex
(Fig. 7A). For this reason, the photolysis was monitored by the
change in the emission spectra. During photolysis the emission
intensity decreased due to the release of histamine and formation
of the non-emissive bis-aquo photoproduct, Fig. 7B,C.
The quenching of luminescence as the photosubstitution of
histamine increases can be used to follow the photochemical
delivery of histamine to specific physiological targets and as a
diagnostic agent.
Biological activity
Solubility and lipophilicity. The experimental solubility (S) in
aqueous buffer tris–HCl solution (pH 7.4 and pH 3.6) and parti-
tion coefficient (log P) in octanol–water solution are listed in
Table 2. The complexes show negative values of log P, indicat-
ing that they are hydrophilic, whereas the uptake experiments,
shown below, indicate their ability to pass through the cell mem-
brane. As expected, there is a direct correlation between the S
and log P values.
In vitro acetylcholinesterase (AChE) inhibition. The role
played by histamine on the cognitive and cholinergic system,
leads us to examine the effect of complexes on the AChE
enzyme activity.20–22 The studies were carried out using the
spectrophotometric method developed by Ellman et al.23
The experimental plots of absorbance of substrate by complex
(1R + 2A) at different concentrations shown in Fig. 8A showed
typical substrate-saturation curves. The inhibition mode of AChE
was studied using the Lineweaver–Burk plot, Fig. 8B. From the
Fig. 4 Luminescence intensity decay (blue line) of 2A in CH3CN. Excitation at 472 nm, and emission at 640 nm. Also shown, the instrument
response function (green line) and curves (red line) from the fit to monoexponential decay (A) and bi-exponential decay (B). The plots of the residuals
are presented for both fits.
Table 2 The time-resolved fluorescence parameters (lifetime τi and
normalized pre exponential factor bi) obtained from the fit of intensity
decay data to biexponential curves, evaluated by χ2red values. The
percentile values in the table give the contribution to the total
fluorescence emission
τ1 (ns) τ2 (ns) b1 b2 χ
2
red %1 %2
2A 137.3 ± 0.1 20.5 ± 0.9 0.904 0.096 1.071 98.4 1.6
1R + 2A 258.1 ± 0.4 35.5 ± 1.5 0.902 0.098 1,142 98.5 1.5
6730 | Dalton Trans., 2012, 41, 6726–6734 This journal is © The Royal Society of Chemistry 2012
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plots, the kinetic parameters: maximum rate constant, (1/rate
versus 1/[substrate]) and the apparent Michaelis–Menten con-
stant (km) were calculated. The values of Km (0.48, 0.25,
0.21 and 0.18 μmol−1 L) for concentrations of 0, 15, 25 and
50 μmol L−1, respectively are indicative of a reversible and
mixed noncompetitive type of inhibition.
These results demonstrated that the complexes do not compete
with the substrate for the active site of the enzyme, but influence
the binding of the substrate with the enzyme inhibiting it.
Probably, the active site inhibitor of complexes and substrate are
close, leading to a change in conformation of the enzyme that
affects the enzyme–substrate interaction.24 The intersection of
the linear plot of 1/rate versus concentration with the x-axis was
able to estimate the inhibitor constant,25 Ki as 38.5 μmol L
−1.
The IC50 (50% AChE inhibitory effect) of (1R + 2A) was deter-
mined to be 21 μmol L−1.
When compared to many commercially proven drugs in
various countries to treat Alzheimer’s disease we can affirm that
this complex is an inhibitor that is nine times stronger than the
commercial drug rivastigmine26 (IC50 = 181.39 μmol L
−1), but it
is much weaker than the drugs huperzine A (IC50 = 0.082 μmol
L−1), donepezil (IC50 = 0.001 μmol L
−1) and galantamine
(IC50 = 0.006 μmol L
−1).
The absence of the inhibitory activity of either free phenan-
throline, free histamine or precursor complex cis-[Ru(phen)2Cl2]
suggests that the coordination of histamine to the fragment
Fig. 5 Continuous photolysis of (1R + 2A) in CH3CN solution at 420 nm light irradiation: (A) change in the absorption spectrum; (B) change in the
emission spectrum; (C) change in the 1H-NMR spectra in the region of the imidazole ring proton, insert: in the region of phenanthroline. Irradiation
times, tirr = 0–20 min.
Fig. 6 Continuous photolysis of 2A in CH3CN solution at 420 nm light irradiation: (A) change in the absorption spectrum; (B) change in the emis-
sion spectrum; (C) change in the 1H-NMR spectra in the region of the imidazole ring proton, insert: in the region of phenanthroline.Fig. 7 Continuous photolysis in aqueous buffer solution (Tris–HCl, pH 7.4) at 420 nm light irradiation for (1R + 2A). (A) Change in the absorption
spectrum in water; (B) change in the emission spectrum in buffer pH 7.4 and (C) change in the emission spectrum in buffer pH 7.4 + BSA.
This journal is © The Royal Society of Chemistry 2012 Dalton Trans., 2012, 41, 6726–6734 | 6731
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[Ru(phen)2]
2+ contributes to the inhibition of enzymes in a
significant way.
In vitro HeLa cells proliferation inhibition. The complexes
were assayed for cell proliferation inhibitory activity with the
human cervical adenocarcinoma cell line HeLa-ATCC CCL-2,
at concentrations of 1, 10, 100, 500, 1000 μmol L−1, Table 3.
The activity profiles of the two complexes show higher differ-
ences between them but they failed to prevent the cellular pro-
liferation of the HeLa cell lines up to the concentration DL50 >
650 μmol L−1, while the sensitivity of the cell line to doperzine
is well known (DL50 = 191 μmol L
−1).27 According to the
description above, free histamine has been shown to be involved
in the proliferation of normal and malignant cells, in particular, it
promotes HeLa cell growth.28–30
Cell uptake studies. The confocal microscopy fluorescent
imaging of fixed HeLa cells with the complexes show that the
complexes are located in the cellular interior, having a stronger
emission (monitored at 660 nm) in the cytoplasm penetrating in
the nuclear region (Fig. 9). These results indicate that the large
size of the complex, its hydrophobicity (high S, small log P) and
its positive charge (+2) does not prevent the complex cell
uptake.
Conclusion
The observance of visible absorption, luminescence and high
photoreactivity towards histamine dissociation combined with
low cytotoxicity and cell uptake into HeLa line cells shows the
potential utility of these complexes as drugs and/or research
tools and promises a more effective delivery of histamine to
specific physiological targets by controlling the irradiation area
and intensities. Our results also offer the opportunity to map the
reactivity of histamine in the physiologic medium using lumine-
scence measurements.
Experimental
General
All reactions were performed under a nitrogen atmosphere.
RuCl3–xH2O, 1,10′-phenanthroline (phen), histamine (hist) and
Fig. 8 (A) Plots of absorbance of substrate by complex (1R + 2A) at different concentrations. (B) Lineweaver–Burk regression of the Michaelis–
Menton plot showing noncompetitive mixed inhibition for (1R + 2A) at concentrations of 0, 15, 25 and 50 μmol L−1.
Table 3 Values of DL50 for HeLa cells, Ki and IC50 for AChE inhibitory effect, solubility and lipophilicity measurements for complexes (1R + 2A)
and 2A
Compound DL50 (μmol L
−1) Ki (μmol L
−1) IC50 (μmol L
−1) Solubility (10−4) pH 3.6/pH 7.4 Lipophilicity pH 3.6/7.4
1R + 2A 925 38.5 21.0 13.6/11.4 −1.41/−1.14
2A 652 37.2 22.3 7.98/6.43 −1.01/−0.962
Fig. 9 Confocal microscopy of HeLa cells with (a) complex 2A and (b) complexes (1R + 2A). Excitation wavelength of 488 nm.
6732 | Dalton Trans., 2012, 41, 6726–6734 This journal is © The Royal Society of Chemistry 2012
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lithium chloride were obtained from Aldrich; tetrabutylammo-
nium perchlorate (TBAP) was obtained from Strem; bovine
serum albumin (BSA) was obtained from Sigma. BSAwas used
without further purification (purity 96% by electrophoresis). The
buffer used was Trizma hydrochloride (Tris/HCl 50 mM), from
Sigma-Aldrich. For the inhibition experiments were used the
reagents: acetylcholinesterase (AChE) 500 UN, acetylthiocholine
iodide and 5,5′-dithiobis(2-nitrobenzoic acid)(3,3′-6) (DTNB)
from Fluka. HPLC grade acetonitrile and dichloromethane were
distilled prior to use. cis-[Ru(phen)2(Cl)2] was prepared accord-
ing to the literature method described for the 2,2-bipyridine
complexes.31
Synthesis of cis-[Ru(phen)2(hist)2](PF6)2·2H2O, complex
(1R + 2A).
cis-[Ru(phen)2Cl2] (0.20 g; 0.25 mmol) was dissolved in a 1 : 1
EtOH–H2O mixture (10 mL), and an amount of histamine
(0.056 g; 0.50 mmol) was added. The solution was stirred under
nitrogen atmosphere for 8 h under reflux. A stoichiometric
amount of NH4PF6 was added to precipitate the complex and the
resultant solution cooled at 0 °C overnight. The reddish-brown
precipitate of cis-[Ru(phen)2(hist)2](PF6)2 was filtered, washed
with water and dried under vacuum (70% yield).
Synthesis of cis-[Ru(phen)2(hist)](PF6)2·2H2O, complex 2A
Complex 2Awas obtained by the reaction of cis-[RuCl2(phen)2]
with 1 equiv. of histamine after adjusting the pH to 10 with the
addition of triethylamine (52.3 μL; 0.37 mmol). The compound,
after isolation as the bis-hexafluorophosphate salt, was filtered,
washed with water and dried under vacuum (60% yield). Anal.
calcd for RuC29H25N7P2F12·2H2O: C, 38.76; H, 3.25; N, 10.91.
Found: C, 39.08; H, 3.32; N, 11.02%.
Spectroscopic techniques
Optical spectra were recorded on an Agilent 8453 UV-vis
spectrophotometer. CHN elemental analyses were performed on
an EA 1110 CHNS-O Carlo Erba Instrument in the Micro-
analytical Laboratory at Universidade Federal de São Carlos
(SP). NMR experiments were carried out in a CD3CN solution
using a BRUKER DRX-400 spectrometer. All chemical shifts
(δ) are given in ppm units with reference to the hydrogen signal
of the methyl group of tetramethylsilane (TMS) as internal stan-
dard and the coupling constants (J) are in Hz.
Monochromatic irradiations at 420 nm were generated either
using a 200 W xenon lamp in an Oriel model 69911 Universal
Arc Lamp source selected with an appropriate interference filter
(Oriel) or a RMR-600 model Rayonet Photochemical reactor
using RMR-4200 lamps. The experiments were carried out at
room temperature in 1.00 cm path length 4 side quartz cells
capped with a rubber septum. The magnetically stirred solutions
(∼10−5–10−2 mol L−1 initial complex concentration) were deoxy-
genated with pure nitrogen. The progress of the photoreaction
was monitored by spectroscopic (UV-vis, luminescence and 1H
NMR) techniques.
Emission spectra were recorded on a Shimadzu RF-5301PC
fluorescence spectrophotometer. Luminescence quantum
yields were relatively measured using a standard φ = 0.062 for
[Ru(bpy)3]
2+ in CH3CN.
32
Time-correlated single-photon counting (TCSP) method was
used to obtain fluorescence emission decay curves.33 The exci-
tation source was a Tsunami 3950 Spectra Physics titanium–
sapphire laser, pumped by a solid state Millenia X Spectra
Physics laser. The repetition rate of the 5 ps pulses was set to 800
kHz using the pulse picker Spectra Physics 3980. The laser was
tuned to give output at 945 nm and a second harmonic generator
LBO crystal (GWN-23PL Spectra Physics) gave the 472 nm exci-
tation pulses that were directed to an Edinburgh FL900 spec-
trometer, where the L-format configuration allowed the detection
of the emission at a right angle from the excitation. The emission
wavelength was selected by a monochromator, and emitted
photons were detected by a refrigerated Hamamatsu R3809U
microchannel plate photomultiplier. The FWHM of the instru-
ment response function was typically 2.20 ns, and measurements
were made using time resolution of 0.245 ns per channel. A soft-
ware provided by Edinburgh Instruments was used to analyse the
decay curves, and theadequacy of the multi-exponential decay
fitting was judged by inspection of the plots of weighted
residuals and by statistical parameters such as reduced chi-square.
Acetylcholinesterase activity
The Michaelis–Menten kinetics were assayed according to the
spectrophotometric method developed by Ellman et al.23 with
minor modifications. A stock solution of 50 μmol L−1 was pre-
pared by dissolving the appropriate amount of the complexes
(1R + 2A) and 2A in methanol. The diluents in a concentration
of 0–25 μmol L−1 were used for inhibition studies. A mixture
containing 100 μL of the complex, 2.9 mL of a solution contain-
ing tris–HCl buffer, 50 mmol L−1 (pH 8.0), DTNB, 333 μmol
L−1, NaCl, 0.1 mol L−1 and MgCl2, 0.02 mol L
−1, and 15 μL of
enzyme solution (0.025 units of AChE, prepared in 15 μmol L−1
solution of BSA in tris–HCl buffer, 50 mmol L−1, pH 8.0) was
incubated for 15 min at room temperature. After this period, the
reaction was initiated with the addition of 10 μL of acetylthio-
choline iodide (25–150 μmol L−1). The hydrolysis of the sub-
strate could be observed by the formation of a yellow compound
(5-thio-nitrobenzoate). Absorbance was measured at 412 nm.
Measurement were made after 5 min of the hydrolysis reaction.
The inhibition type was justified by the linear Lineweaver–Burk
equation. The constant parameters Km and Vmax were evaluated
by the linear regression of the reaction (1/rate) versus the sub-
strate concentration. The value of Ki was then obtained from the
intersection of the linear plot of (1/rate) versus complex concen-
tration with the x-axis.
Solubility
Solubility of complexes (1R + 2A) and 2A in buffers tris–HCl,
50 mmol L−1, pH 3.6 and 7.4 were determined at 37.0 ± 0.5 °C.
These experiments were carried out by adding an appropriate
amount of complex until saturation in 2 mL of buffer solution.
Suspensions were shaken for 24 h at 50 rpm until equilibrium
was attained. Samples were centrifuged for 8 min in a centrifuge
at 220 rpm. The concentration of the complex in the filtrate was
determined by UV-vis spectrophotometer.
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Lipophility
Lipophility was determined by partitioning octanol and buffer
tris–HCl, 50 mmol L−1. The complex (100 ppm) was added
to a solution containing 1 mL of octanol and 1 mL of buffer.
Solutions were then placed in the shaker for 24 h at 50 rpm.
Samples were centrifuged for 8 min at 220 rpm. The solutions
were separated from the two phases and the concentration of the
complex was determined in the organic and aqueous phases by
ICP-OES (Perkin Elmer Optima 3000DV). The experiments
were done in triplicate. Equation log P = log(Co/Cw) was used to
calculate log P: where Co and Cw are the molar concentrations of
complex in octanol and aqueous phases, respectively.
Cell culture
HeLa cells were maintained in Dulbecco’s Minimum Eagle
medium (DMEM) supplemented with 10% fetal calf serum
(FCS) and 1% penicillin/streptomycin, at 37 °C in a humid incu-
bator with 5% CO2. In order to detach cells from the bottle,
trypsin–EDTA solution was used.
Cytotoxicity
HeLa cells were seeded in 24 well plates at the initial density of
5 × 104 cells per well. 18 h after plating, the cells were exposed
to the complexes (1, 10, 100, 500 and 1000 μmol L−1 in DMEM
without phenol red and 1% FCS) for 3 h. The medium was then
removed, cells were washed with PBS, and fresh medium was
added. The MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium) was performed 48 h after. Cells were incubated
with 0.25 mg mL−1 MTT for 2 h. Formazan crystals were solu-
bilized by adding 500 μL of DMSO. Absorbance was measured
at 550 nm and the values of cell survival are presented as
percent of control.
Confocal microscopy
HeLa cells were seeded in individual plates containing a glass
coverslip (1 × 104 cells per well). 18 h after, cells were exposed
to the complexes (3.49 × 10−4 mol L−1 in DMEM without
phenol red and 1% FCS) for 4 h. The cells were then washed
with PBS and the fluorescence images were obtained in a con-
focal microscope (Zeiss LSM510), using a set of filters with
excitation at 488 nm and emission from 600–700 nm.
Acknowledgements
The authors would like to acknowledge FAPESP (Proc 2009/
08218-0), CNPq (Universal 470890/2010-0) and CAPES for the
grants and fellowships given to this research.
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